In the last year, our work has covered several major areas that build on our previous work: I. Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 20 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling, cytokine production and cytotoxicity, via effects on activation of PLC-g, Ca++ mobilization, ERK and mTOR (Shaeffer et al. Science 1999; Shaeffer et al 2000, 2001, Nature Immunol.; Broussard et al. Immunity 2006; Horai et al. Immunity 2007; Gomez-Rodriguez et al., Immunity 2009, JEM 2014, Nature Comm 2016; Kapnick et al, J. Immunol. 2017, PNAS 2017). Confirming its importance, mutations of Itk have been described in a profound immunodeficiency associated with EBV-induced lethal lymphoproliferation. To follow up on previous observations that Itk is required for Th2- and Th9-mediated responses, such as those associated with hypersensitivity and asthma (Gomez-Rodriguez, Nature Comm 2016; Schaeffer et al, Nature Immunol 2001), we have now developed a series of conditional Itk-mutant mice, including ones that can be inhibited acutely by designer inhibitors (Gomez-Rodriguez, in progress), providing potential models for Itk-based therapeutics. II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work initially focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014; Cannons et al, Front Immunol 2018). To further understand these defects, we generated a mouse model that recapitulates multiple features of the disease, and have used these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. In the last year, we published work dissecting T and B cell-intrinsic and T cell-extrinsic components to these phenotypes, including autoimmunity, and have revealed a major role for the commensal microbiome in the development of autoantibodies (Preite et al. Nature Immunol. 2018, Front Immunol 2019). Our more recent work has revealed a major role for PI3K in the development of T cell memory as well as responses to chronic infection (Cannons et al, Front Immunol. 2018 Cannons et al, in progress). III. Regulation of Tfh cells and humoral immune responses: Another major focus of our work was SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1),characterized by fatal EBV-infection, lymphomas, and antibody defects (Cannons et al. J. Immunol 2017; Panchal et al, Frontiers Immunol., 2018). Using gene-targeted mice we generated (Czar et al 2001), we previously showed that SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination (Crotty et al. Nature 2004; Cannons et al. 2006). Our work has provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells (Qi et al, Nature 2008; Cannons et al, Immunity 2010; Lu et al, Immunity 2011), which are the critical helper T cell population providing signals to B cells for germinal center formation and long-term humoral immunity, required for an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). Stemming from this work, we have continued to explore genes important for Tfh cell differentiation and function, including the transcription factor TCF1, a component of the Wnt signaling pathway, that is selectively expressed and required for Tfh cells in response to viral infection (Wu et al, Cell Reports, 2015). To further uncover genes important for Tfh differentiation, we are using CRISPR-mediated mutagenesis in primary mouse T cells from Cas9 mice (Huang et al Curr Prot Immunol 2019) to screen for genes affecting Tfh cells formation in vivo, as well as T cell adhesion in vitro (which is required for T cell interactions with other cells). IV. In recent work, we have found that similar signaling and transcriptional networks are required for long-term CD8 cell responses to chronic infection, and that TCF1 marks a population of stem-like CD8 cells with remarkable transcriptional and metabolic overlap with Tfh cells (Wu et al, Sci Immunol 2016). These stem- or progenitor-like cells are now recognized as critical for maintaining responses to chronic infection and cancer and are the cells that are responsible for responses to checkpoint blockade therapies. In the last year, we have used single-cell RNAseq methods to learn more about this population during chronic infection and their differences from memory cells during acute infection. We have now identified another transcription factor, TOX, that is critical for the maintenance of T cells during chronic infections as well as cancer (Yao et al Nature Immunol 2019). Work from our lab, as well as 5 other groups published concurrently in Nature, Immunity, PNAS and Hepatology, has now revealed that TOX promotes expression of factors associated with exhaustion, but which are critical to allow T cells to persist during chronic infection. This work has important implications for understanding and potentially manipulating T cell responses to chronic infections such as HIV and HCV, as well as cancer. IV. To increase our ability to probe the immune system, we have developed new CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al, PLOS One, 2016 ; Huang et al, Curr Protoc Immunol 2019). We are using these tools to identify and probe function of genes involved in Tfh cell differentiation, uncovering a role for the VHL-HIF1a axis (Huang et al, in preparation). In a related set of studies, we have identified a new regulator pf lymphocyte adhesion (Johansen et al, In progress). V. We have continued to collaborate with others on generation of new mouse models of human genetic diseases, including L. Notarangelo and L. Biesecker (Lindhurst et al, Hum Mol Genet 2019) and on immunological and signaling characterization of mouse models (Kanellopoulou et al, JEM 2019; Collins et al, Cell, In Press).